Clostridium difficile associated disease (CDI) is an important cause of morbidity in hospital and nursing home patients, and is being increasingly recognized as an important cause of mortality. Estimates put the economic burden of CDI at 1-3 billion dollars in the U.S. alone. The overall goal of this project is to develop an anti-toxin humn monoclonal antibody product for prevention and/or treatment of C. difficile infection. Due to specificity, stability, safety, and the specific targeting of toxins (as opposed to selective pressre on the bacterium), a mAb product is ideal for prevention and treatment of the diseases caused by C. difficile. Antibodies are the only category of FDA-approved therapeutic that specifically neutralize toxins. We have isolated a panel of neutralizing human monoclonal antibodies (mAbs) that recognize the toxins of C. difficile. In terms of their neutralizing potency in vitro, these mAbs are superior to mAbs previously reported that are in clinical development [1]. Preliminary testing in vivo has demonstrated protection in the hamster model that was superior to vancomycin. We will produce each of our human mAbs against C. difficile toxins A and B (TcdA, TcdB) in a scalable production system that can provide antibodies for therapeutic or prophylactic indications. Each mAb, as well as the combination of mAbs, will be evaluated for effectiveness in preventing and treating C. difficile infection in the hamster model to select the lead mAb cocktail for continued development. Specific Aim #1. Produce quantities of each anti-TcdA and anti-TcdB human mAb for in vitro characterization and animal testing in a novel manufacturing system. Human anti-TcdA and anti-TcdB mAbs have been generated using transgenic mice containing human immunoglobulin loci (HuMab-Mouse(r)). Three anti-TcdA and three anti-TcdB mAbs have been developed under license by Mapp. The mAbs all exhibited potent neutralizing activity and have subsequently been produced in a scalable plant-based system. Combined, these mAbs are potent immunoprotectants in the hamster challenge model. Specific Aim #2. Evaluate individual mAbs and combinations of mAbs in vivo for prevention and treatment of CDI. MAbs produced in Specific Aim #1 will be compared in the hamster model of C. difficile infection. The anti-TcdA mAbs will first be evaluated individually to determine the baseline of protection. Only anti-TcdA has been observed to provide significant protection alone, whereas anti-TcdB may extend the duration and effectiveness of anti-TcdA protection [1]. The best anti- TcdA mAb will then be further tested in combination with each anti-TcdB mAb to determine the synergistic potential of mAb pairs. The best mAb pair will be selected for further development in pre-clinical and clinical protocols largely funded by a Phase II SBIR grant. PUBLIC HEALTH RELEVANCE: The efforts in this proposal will help in the development of a drug product for preventing and/or treating Clostridium difficile infection (CDI), a cause of significant morbidity and mortality in hospitals and nursing homes. The economic cost of CDI has been conservatively estimated at $1-3 billion per year in the U.S. The recent characterization of an epidemic C. difficile strain with increased virulence and antibiotic resistance has made the development of alternative therapies even more pressing.